Method for Monitoring Heptadecanoic Acid in Human and Animal Samples

ABSTRACT

Methods for detecting risks for and/or causes of metabolic syndrome or hyperferritinemia in accordance with several embodiments can include the step of measuring the level of heptadecanoic acid in a blood sample of a subject. The methods of several embodiments can further include the step of deeming the subject as having or being at risk of metabolic syndrome if the amount of heptadecanoic acid is below 0.4% of all fatty acids in the sera or plasma. The methods for treating metabolic syndrome or hyperferritinemia according to several embodiments can also include the step of administering a daily dose of heptadecanoic acid to a subject suffering from metabolic syndrome or hyperferritinemia for a period of time from three weeks to twenty-four weeks, wherein the minimum daily dose comprises about 3 mg per lb (or 6 mg per kg) of body weight.

This application is a divisional application of U.S. patent applicationSer. No. 14/591,660, filed Jan. 7, 2015 by Stephanie Venn-Watson, for aninvention entitled “Use of Heptadecanoic Acid (C17:0) To Detect Risk OfAnd Treat Hyperferritinemia and Metabolic Syndrome”. The '660application is assigned to the same assignee as the present invention.

FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

The United States Government has ownership rights in this invention,pursuant to passing of title to a Subject Invention under Federal GrantN00014-12-1-0294 (National Marine Mammal Foundation). Licensinginquiries may be directed to Office of Research and TechnicalApplications, Space and Naval Warfare Systems Center, Pacific, Code72120, San Diego, Calif., 92152; telephone (619) 553-5118; email: sscpac t2@navy.mil, referencing NC 103857.

FIELD OF THE INVENTION

The present invention pertains generally to the detection and treatmentof metabolic syndrome and hyperferritinemia. More particularly, thepresent invention pertains to the detection and treatment of metabolicsyndrome and hyperferritinemia by measuring and raising (if necessary)the level of heptadecanoic acid in the blood of a subject.

BACKGROUND OF THE INVENTION

There is a worldwide pandemic of diabetes in humans. In addition todiabetes, more than one in every three adults in the United States, anestimated 86 million people have metabolic syndrome, also calledprediabetes. An estimated 440 million people in the world will possiblyhave diabetes by 2030, and there is a dire need to stem concurrentpandemics of metabolic syndrome, type 1 diabetes and type 2 diabetes.

In human subjects, high serum ferritin and iron overload have also beenassociated with metabolic syndrome, type 2 diabetes, and type 1diabetes. While iron overload is most commonly associated with amutation in the HFE gene resulting in C282Y substitutions, there isincreasing recognition of high serum ferritin that is not associatedwith known genetic mutations. Ferritin is a measurement of total ironbody stores. High ferritin in the blood (i.e. hyperferritinemia) andassociated iron overload have been associated with metabolic syndrome(prediabetes), type 2 diabetes and type 1 diabetes in humans. Until now,serum ferritin has not been routinely tested in human subjects, but thefew screening studies known in the prior art have demonstrated that asurprisingly high percent (28% and 12%) of healthy elderly men and womenin the United States have hyperferritinemia. It is unknown precisely howhigh ferritin increases the risk of diabetes, but proposed mechanismsinclude direct injury to the liver and pancreas from excessivedeposition or indirect injury from increased oxidative radicals.

Like human subjects, bottlenose dolphin (Tursiops truncatus) subjectscan also be susceptible to metabolic syndrome, including high insulin,glucose, triglycerides, fatty liver disease, and iron overload. Ironoverload in dolphins, involving excessive iron deposition primarily inthe liver's Kupffer cells, can be progressive with age and can beassociated with elevated insulin, lipids, and liver enzymes. Thisdisease is associated with neither mutations in the HFE gene norincreases in studied acute phase proteins. Similar to humans, ironoverload in dolphins is treated with phlebotomy, and repeated treatmentsare needed throughout life due to returning elevations of serumferritin. The underlying causes of iron overload and hyperferritinemiain dolphins are unknown.

Dolphins at the Navy Marine Mammal Program (MMP) are a well-studieddolphin population with regard to metabolic syndrome, and thispopulation has a higher risk of developing metabolic syndrome whencompared to wild dolphins, such as wild dolphins living in Sarasota Bay,Fla., for example. When comparing the two populations, neither body massindices nor stress indices (i.e. cortisol) are risk factors formetabolic syndrome in MMP dolphins. In studies comparing values ofblood-based indicators of metabolic syndrome, MMP dolphins have beenolder than Sarasota Bay dolphins; older age of the MMP dolphinpopulation is further supported by its higher annual survival rates andlonger lives compared to wild dolphins, including those living inSarasota Bay. Proposed risk factors for metabolic syndrome in dolphinscan include advanced age, differences in feeding and activity schedules,and differences in dietary fish. It can be hypothesized that differencesin dietary fish (and certain fatty acids associated with particulartypes of fish) can be responsible for differences in the risk ofmetabolic syndrome and iron overload in dolphins.

In view of the above, it is an object of the present invention toprovide a method for detecting protective and risk factors against andfor metabolic syndrome and hyperferritinemia in mammal subjects such asdolphins and humans. Another object of the present invention is toprovide a method for treating metabolic syndrome and/orhyperferritinemia in mammal subjects, such as dolphins and humans. Stillanother object of the present invention is to provide a method fordetecting metabolic syndrome and/or hyperferritinemia in mammalsubjects, such as for dolphins and humans that increases the level ofheptadecanoic acid of the subject sera. Yet another object of thepresent invention is to provide a method for detecting and treatinghyperferritinemia without resorting to phlebotomy. Another object of thepresent invention is to provide a method for detecting and treatingmetabolic syndrome and/or hyperferritinemia in mammal subjects, such asdolphins and humans that is easy to accomplish in a cost-effectivemanner.

SUMMARY OF THE INVENTION

Methods for detecting risk factors for metabolic syndrome orhyperferritinemia in accordance with several embodiments can include thestep of measuring the level of heptadecanoic acid (also called C17:0 ormargaric acid) in a blood sample of a subject. The methods of severalembodiments can further include the step of deeming the subject ashaving risk factors for or causes of metabolic syndrome if the amount ofheptadecanoic acid represents below 0.4% of the total fatty acidspresent in the serum or plasma. The methods for treating metabolicsyndrome or hyperferritinemia according to several embodiments can alsoinclude the step of administering a daily dose of heptadecanoic acid toa subject suffering from metabolic syndrome or hyperferritinemia for aperiod of time from three weeks to twenty-four weeks, with continuousdaily doses thereafter to prevent recurrence, wherein the total dailydose comprises an approximate minimum of 3 mg heptadecanoic acid per lb(or 6 mg heptadecanoic per kg) of mammal body weight. The administrationof heptadecanoic acid according to several embodiments can also treathyperferritinemia without requiring phlebotomy.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the present invention will be best understood fromthe accompanying drawings, taken in conjunction with the accompanyingdescription, in which similarly-referenced characters refer tosimilarly-referenced parts, and in which:

FIGS. 1-4 are graphs that illustrate significant, inverse linearassociations between heptadecanoic acid (as % serum fatty acids) andinsulin, glucose, triglycerides, and ferritin, respectively, usingsimple linear regression models;

FIG. 5 is a comparison of sera heptadecanoic acid levels between a casestudy population of subjects that are highly susceptible tohyperferritinemia, iron overload, and metabolic syndrome (elevatedinsulin, glucose, and triglycerides) and a control study population withlow susceptibility to hyperferritinemia and metabolic syndrome;

FIG. 6 is a bar graph that illustrates the amount (mg/100 g) ofheptadecanoic acid in a variety of fish types and dairy products;

FIG. 7 is a bar graph that illustrates the comparison of total averagedaily dietary intake of heptadecanoic acid in the original versusmodified diet for the feeding study supporting the claimed inventionaccording to several embodiments;

FIGS. 8-9 are graphs that illustrate the effects of the diet depicted inFIG. 7 on an increasing heptadecanoic acid in both subject sera (%) andsubject red blood cell (RBC) membranes (μg/ml) during a 24-week feedingstudy;

FIG. 10 is a graph that illustrates the effects of the diet on serainsulin levels for the subjects of the feeding study;

FIGS. 11-12 are graphs that illustrate the effects of the diet depictedin FIG. 7 on a decreasing sera ferritin levels during a 24-week feedingstudy;

FIGS. 13-14 are graphs that illustrate the effects of the diet on seraglucose, and triglyceride levels for the subjects of the feeding study;

FIG. 15 is a scatter plot of heptadecanoic acid (% serum fatty acids)versus insulin in both case and control populations, which indicatestherapeutic thresholds of heptadecanoic acid for the present inventionaccording to several embodiments; and,

FIG. 16 is a scatter plot of heptadecanoic acid (% serum fatty acids)versus ferritin, which indicates therapeutic thresholds of heptadecanoicacid for the present invention according to several embodiments.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Serum Fatty Acids and Metabolic Syndrome Indices

Table 1 is a table of blood values of blood samples that were taken froma managed population of thirty dolphins from the Navy Marine MammalProgram (MMP). Dolphins at the Navy Marine Mammal Program (MMP) are awell-studied dolphin population with regard to metabolic syndrome, andthis population has a higher risk of developing metabolic syndrome whencompared to wild dolphins, such as wild dolphins living in Sarasota Bay,Fla., for example. 2 h postprandial blood values from MMP dolphins withelevated insulin (Elevated insulin levels were defined as values greaterthan or equal to the 75th quartile among the 30 Group A dolphins (15μIU/ml), n=8) were compared to MMP dolphins without elevated insulin(n=22). Table 1 illustrates values of elevated versus non-elevatedinsulin. There were no differences in groups with regard to age (30±7and 25±14 years, respectively; P=0.32) or sex (percent female 37.5% and54.6%, respectively; P=0.68). Similar to what has been previouslyreported with MMP dolphins, those with elevated insulin were also morelikely to have higher glucose, triglycerides, and gamma-glutamyltranspeptidase (GGT) when compared to MMP dolphins with non-elevatedinsulin, which can support the proposition that dolphins with elevatedinsulin represent those with or at higher risk of metabolic syndrome.

TABLE 1 Elevated Non-elevated insulin insulin Metabolic variable (n = 8)(n = 22) P value Metabolic panel Glucose (mg/dl) 114 ± 7  100 ± 8  0.002Triglycerides (mg/dl) 164 ± 205 128 ± 43  0.007 Gamma-glutamyl 33 ± 1224 ± 10 0.046 transpeptidase (U/l) Iron (μg/dl) 178 ± 39  177 ± 63  0.64Ferritin (ng/ml) 5,931 ± 4,210 3,131 ± 3,371 0.13 Transferrin saturation(%) 53 ± 14 57 ± 21 1.0 HbA1c (%) 5.1 ± 0.2 5.2 ± 0.4 0.78 Estimatedaverage glucose 85 ± 7  86 ± 12 0.78 (mg/dl) Serum fatty acid (%)Heptadecanoic acid (C17:0) 1.0 ± 0.2 1.6 ± 0.3 0.0008 Oleic acid(C18:1n9) 21 ± 2  18 ± 4  0.03 Linoleic acid (C18:2n6) 1.6 ± 0.1 1.3 ±0.2 0.03 Arachidonic acid (C20:4n6)   3 ± 0.3 4 ± 1 0.004Eicosapentaenoic acid 10 ± 1  13 ± 3  0.006 (C20:5n3) Myristic acid(C14:0) 1.7 ± 0.4 1.9 ± 0.6 0.39 Palmitic acid (C16:0) 14 ± 1  14 ± 2 0.25 Palmitoleic acid (C16:1n7) 6 ± 1 6 ± 1 0.17 Stearic acid (C18:0) 12± 2  11 ± 2  0.66 Vaccenic acid 6 ± 2 6 ± 1 0.73 (C18:1cis-11n7)α-Linolenic acid (C18:3n3) 0.2 ± 0.1 0.4 ± 0.7 0.28 Erucic acid(C22:1n9) 4.7 ± 1.7 4.6 ± 1.3 0.87 Docosatrienoic acid (C22:3n3) 0.1 ±0.1 0.1 ± 0.1 0.47 Docosapentaenoic acid 1.9 ± 0.1 2.0 ± 0.3 0.98(C22:5n3) Docosahexaenoic acid 9.2 ± 0.9 8.9 ± 1.2 0.34 (C22:6n3)Tricosylic acid (C23:0) 0.5 ± 0.1 0.5 ± 0.3 0.32 Nervoic acid (C24:1n9)1.0 ± 0.5 0.9 ± 0.4 0.76

From Table 1 above, it can be seen that dolphins with elevated insulinalso had higher oleic acid and linoleic acid; and lower heptadecanoicacid, arachidonic acid, and EPA compared to non-elevated insulindolphins. Thus, the methods of the present invention can focus on thesefive fatty acids, and margaric acid in particular (in the specification,heptadecanoic acid, margaric acid and C17:0 shall be taken to mean thesame thing). The complete set of data can be found in the paper byStephanie Venn-Watson et al. entitled “Reversion of Hyperferritinemiaand Prediabetes with Dietary Margaric Acid”, which is included inApplicant's Invention Disclosure Statement. This paper is incorporatedby reference into this application. The manner in which the serum andred blood cell fatty acid profiles was accomplished is described morefully in a paper by Susan A. Lagerstedt et al. entitled “QuantitativeDetermination of Plasma C8-C26 Total Fatty Acids for the BiochemicalDiagnosis of Nutritional and Metabolic Disorders”.

Referring now to FIGS. 1-4, FIGS. 1-4 are plots 10, 12, 14, and 16 ofmargaric acid (as a percentage of serum fatty acids in sera) versus 2 hpostprandial insulin (μIU/ml), glucose (mg/dl), triglycerides (mg/dl)and ferritin (ng/ml), respectively, for the 30 MMP dolphins cited above.For each of the respective plots 10, 12, 14 and 16 in FIGS. 1-4,respective linear regressions 18, 20, 22 and 24 of the data wereaccomplished. One way to determine heptadecanoic acid in dolphin plasmais described in “An analytical method for Bruce Babson et al.(MicroConstants, Inc.), “Method for the Determination of HeptadecanoicAcid in Dolphin Plasma Using High-Performance Liquid Chromatography withMass Spectrometric Detection”, dated 6 Oct. 2015. The Babson method ishereby incorporated by reference into this specification.

The statistical analyses depicted in FIGS. 1-4 were conducted using theWorld Programming System (World Programming Ltd., Hampshire, UnitedKingdom). Age, sex, and blood values (glucose, HbA1c, estimated averageblood glucose, triglycerides, GGT, iron, transferrin saturation,ferritin, and percent serum fatty acids) were compared between dolphinswith and without elevated insulin. Sex distribution was compared using aMantel-Haenzsel Chi-square test. Age and blood variable values werecompared using a Wilcoxon rank-sum test. For the five fatty acids thathad significant differences between dolphins with and without elevatedinsulin (heptadecanoic acid, oleic acid, linoleic acid, arachidonic acid(AA), and eicosapentaenoic acid (EPA)), simple linear and stepwisemultivariate regressions were used to test for associations betweenthese potential fatty acid predictors and dependent metabolic syndromeindices (insulin, glucose, triglycerides, and ferritin). In allanalyses, significance was defined as a P value less than 0.05.

From FIGS. 1-4, and using the above criteria, it can be seen that amongthe 30 MMP dolphins, percent serum heptadecanoic acid had negativelinear associations with insulin, glucose, triglycerides, and ferritin,respectively. Using the best fit, stepwise regression described above,it can be inferred from FIGS. 1-4 that heptadecanoic acid can be anindependent predictor of insulin (FIG. 1, P=0.0004), glucose (FIG. 2,P=0.0002) triglyceride (FIG. 3, P=0.0004), and ferritin (FIG. 4,P<0.0001) levels.

From the data above, it can be appreciated that there is a linearrelationship between levels of heptadecanoic acid and insulin, glucose,triglycerides and ferritin levels in sera for the MMP dolphins. Toconfirm this appreciation, the margaric acid levels of the sera incontrol population B (the Sarasota Bay dolphins) were checked.

FIG. 5 illustrates the results of the above heptadecanoic acid check.From FIG. 5, it can be seen that the control population of Sarasota Baydolphins had three times the level of heptadecanoic acid (measured as apercent serum fatty acid) than the case population A of MMP dolphins.Table 2 below illustrates a comparison of the blood samples of theabove-cited 30 MMP dolphin versus the sera of 19 wild dolphins in theirnatural habitat (Sarasota Bay dolphins). MMP dolphins were older thanSarasota Bay dolphins (mean age±SD=25.6±12.2 and 12.7±9.0 years,respectively; P=0.002). As shown in Table 2, MMP dolphins had higherinsulin, glucose, triglycerides, ferritin, iron, and transferrinsaturation compared to Sarasota Bay dolphins. MMP dolphins had lowerserum heptadecanoic acid when compared to Sarasota Bay dolphins. Whilered blood cell fatty acids were not collected on the initial group of 30MMP dolphins, this measurement was included for Sarasota Bay dolphins touse as a reference during the subsequent feeding study with MMPdolphins, as described more fully below.

TABLE 2 MMP Sarasota Bay Blood-based variable (n = 30) (n = 19) P valueMetabolic variable Total insulin (μIU/ml) 11 ± 12 2 ± 5 <0.0001 Glucose(mg/dl) 117 ± 10  104 ± 15  0.005 Triglycerides (mg/dl) 148 ± 59  78 ±26 <0.0001 Gamma-glutamyl transpeptidase 27 ± 11 20 ± 6  0.02 (U/L)Ferritin (ng/ml) 3,878 ± 3,754 219 ± 184 <0.0001 Iron (μg/dl) 177 ± 57 109 ± 48  <0.0001 Transferrin saturation (%) 56 ± 20 33 ± 11 <0.0001Targeted serum fatty acid (μg/ml) Heptadecanoic acid (C17:0) 9 ± 2 25 ±9  <0.0001

From the above Table 2 data, it can be seen that among Sarasota Baydolphins, serum heptadecanoic acid (μg/ml) was inversely associated withferritin (R2=0.29 P=0.02). All Sarasota Bay dolphins with ferritingreater than 219 ng/ml (this population's 50th quartile) had serumheptadecanoic acid levels less than 25 μg/ml, suggesting that serumheptadecanoic acid lower than 25 μg/ml may result in an increased riskof hyperferritinemia.

With a renewed focus on heptadecanoic acid, and referring now to FIG. 6,comparisons of diets of MMP dolphins (Case Population A) with diets ofSarasota Bay dolphins (Control Population B) were accomplished todetermine the levels of heptadecanoic acid in the food being eaten bythe two populations. As shown in FIG. 6, capelin, and the primary fishtype fed to MMP dolphins, had no detectable heptadecanoic acid comparedto other fish types. With the exception of squid (not shown in FIG. 6),capelin also had the lowest levels of iron compared to the other fishtypes. As shown in FIG. 6, mullet, and pinfish (which are representativeof fish eaten by Sarasota Bay dolphins), had relatively high levels ofheptadecanoic acid. Mullet and pinfish also had the highest iron levelsamong the fish tested. Due to the known presence of heptadecanoic acidin dairy products, heptadecanoic acid levels were measured inoff-the-shelf dairy products. Dairy products consumed by human are alsoshown in FIG. 6, for comparison. The content of heptadecanoic acid(mg/100 g), from highest to lowest, was 42 (butter), 31 (whole fatyogurt), 19 (whole fat milk), and 10 (2% fat milk). Heptadecanoic acidwas not detected in either nonfat milk <2 mg/100 g or nonfat yogurt <10mg/100 g.

From the above data, it can be seen that there is a linear relationshipbetween heptadecanoic acid and triglycerides, glucose, insulin andferritin in sera, which has been confirmed with measurements of bothheptadecanoic acid in dolphins for both a case population and a controlpopulation, as well as a measure of heptadecanoic acid in the dietseaten by the respective populations.

Building on the above results, a 24-week feeding study was accomplishedon the case population (MMP) dolphins, to determine if theabove-referenced triglycerides, glucose, insulin, and ferritin seralevels could be manipulated by manipulating the C17:0 sera levels. To dothis, the diets of the case population MMP dolphins were manipulated.More specifically, the diets of six MMP dolphins were modified todecrease capelin and introduce pinfish or mullet (fish with an increasedamount of margaric acid) to their diet while maintaining the same dietcaloric intake. Stated differently, and as shown in FIG. 7, the averagedaily intake of heptadecanoic acid was increased from approximately 400mg to 1700 mg. The increase to 1700±500 mg daily heptadecanoic acid wasequal to an approximate minimum daily heptadecanoic acid intake of 3mg/lb body weight (6 mg/kg body weight). It should be appreciatedhowever, that more research is needed to determine the exact minimumdaily amount, and the minimum daily amount may go down or up accordingto future research. To evaluate potential confounding effects of theenvironment outside of the feeding study on the dolphins, eight MMPdolphins, which were housed in the same environment but not included inthe feeding study, were monitored as references; these dolphins also hadroutine monthly blood samples collected during months 0, 1, 3, 4, and 6.

FIGS. 8 and 9 are graphs of sera heptadecanoic acid (as a percent serumfatty acid and RBC in μg/ml, respectively) for the MMP dolphinsresulting from the above-described feeding study. Additionally, meanlevels of margaric acid in Sarasota Bay dolphins (indicated by lines 82and 92 in FIGS. 8-9) are included as a comparison. As can be seen inFIG. 8-9, as a result of the increase in heptadecanoic acid intake,serum levels of heptadecanoic acid were higher in feeding study dolphinsduring weeks 3, 6, 12, 18, and 24 when compared to week 0.

To determine the effects of increased sera heptadecanoic acid depictedin FIGS. 8-9, the insulin in the feeding study dolphins was measured.The measurement results are depicted in FIG. 10. As shown in FIG. 10,the insulin levels of the feeding study dolphins decreased during theperiod of the feeding study, which confirms the effects of the increasedmargaric acid in the subject sera. In addition, and perhaps just asimportantly, a normalization of spread of insulin values for the subjectsera was observed from an initial spread illustrated by line 102 at thestart of the study (0 weeks) to a final spread 104 at 24 weeks.

To determine the effects of increased sera margaric acid depicted inFIGS. 8-9 on ferritin levels, and referring now to graphs 110 and 120 inrespective FIGS. 11 and 12, the ferritin in the feeding study dolphinswas measured. As shown in FIGS. 11 and 12, serum ferritin levelscontinually decreased in all six dolphins throughout the feeding study,with weeks 3 through 24 having lower levels than week 0. Excluding thetwo extremely high ferritin outliers depicted by lines 112 and 114 inFIG. 10 (ferritin levels in the upper thousands to tens of thousands);the remaining dolphins (represented by lines 122, 124, 126 and 128 inFIG. 11) had the lowest mean serum ferritin (243±58 ng/ml) by week 24.Moreover, the mean ferritin levels for these dolphins approached theSarasota Bay dolphins' mean value of 219±184 ng/ml, as depicted by line130 in FIG. 11 (For purposes of the specification a therapeutic levelcan be defined as the mean level of Sarasota Bay dolphins). Due to thedramatic decrease in serum ferritin in all six feeding study dolphins,indices of acute inflammation (ceruloplamsin and haptoglobin) wereassessed. Despite decreases in ferritin, there were no differences inthese two proteins during any of the study weeks compared to week 0,supporting a conclusion that the decreased ferritin was not due todecreased acute inflammation.

In addition to the decrease in ferritin, and referring now to FIGS. 10,13 and 14, there was a distinct decrease in measures of spread forinsulin, glucose, and triglycerides that trended from weeks 0 to 24,i.e., there was a normalization of insulin, glucose and triglycerideslevel in subject sera. Changes in serum insulin, glucose, triglycerides,and ferritin during week 0 were compared to weeks 3, 6, 12, 18 and 24values were compared to week 0 using pairwise comparison t-tests (in thereference population, month 0 values were compared to months 1, 3, 4 and6). Given the apparent tightening or normalization of glucose,triglycerides, and insulin (5 of 6 dolphins) values among feeding studydolphins by week 24, measures of spread (standard deviation, SD, andcoefficient of variance, CV) were compared between weeks 0 and 24 forglucose, triglycerides, and insulin; outcomes were compared to thereference dolphin group. CV was calculated as follows: standarddeviation÷mean).

With regard to decreasing measures of spread, the insulin standarddeviation (FIG. 10) decreased from about 50 to about 12 μIU/ml, whilethe standard deviation for glucose (FIG. 13) decreased fromapproximately 70 to 20 mg/dl. The standard deviation for triglycerides(FIG. 14) decreased from about 200 to 90 mg/dl. The coefficient ofvariation (C.V.) from week 0 to week 24 decreased from 22% to 6% forglucose and 61% to 24% for triglycerides. When limiting to five studydolphins (excluding the outlier sixth dolphin), the insulin C.V.decreased from 100% to 38%. The decrease in measures of spread for thesethree key variables (normalization) is visually apparent from lines 102and 104 in FIG. 10, initial spread 132 and final spread 134 in FIG. 13,and respective initial and final spreads 142 and 144 in FIG. 14.

Among the reference dolphin group for the feeding study (dolphins whosediet was not modified), there was not a difference in serum ferritin(week 0=4,116±2,822 ng/ml) compared to weeks 3, 12, and 18 (4,433±3,000,4,055±2,534, and 3,418±2,059 ng/ml respectively; P=0.43, 0.92, and0.37). There were also no differences in glucose and triglycerides whencomparing week 0 with weeks 3, 12, and 18 (not shown); and the measuresof spread for glucose and triglycerides did not decrease from week 0 toweek 18 (standard deviation=16 and 15 mg/dl, C.V.=16% and 15% forglucose; and 58 and 48 mg/dl, C.V.=74% and 66% for triglycerides,respectively). Similarly, serum heptadecanoic acid (9.3±4 versus 9.3±4ng/dl; P=0.98) glucose, and triglycerides did not differ in mean ormeasures of spread for the reference population when comparing month 0with month 4.

While not statistically significantly different using the appliedmethods based upon the mean, mean levels of all three indicators ofmetabolic syndrome did trend down; for week 0 versus week 24, meaninsulin decreased from 24 to 16 μIU/ml, glucose from 105 to 95 mg/dl,and triglycerides from 132 to 87 mg/dl. In sum, FIGS. 10-14 can be takento mean that increased levels of heptadecanoic acid can result in adecrease of ferritin levels and a normalization of metabolic syndromebiomarkers in subject sera.

This is the first report of heptadecanoic acid as an independentpredictor among a full suite of metabolic syndrome indices, includingglucose, insulin, triglycerides, and associated ferritin. Importantly,when dolphins with hyperferritinemia increased their dietary intake ofheptadecanoic acid by changing fish types fed, ferritin, glucose,triglycerides, and insulin normalized by week 24. Becausehyperferritinemia in humans is associated with metabolic syndrome, andresolution of iron overload with phlebotomy improves insulin resistance,this study may provide important insight into how heptadecanoic aciddeficiencies may be an underlying and treatable cause ofhyperferritinemia and subsequent metabolic syndrome in humans.

Heptadecanoic acid (C17:0), also called margaric acid, is a saturatedfatty acid present in bovine milk fat and was the original component ofmargarine (hence, margarine's name) in the late 1800s. Heptadecanoicacid in margarine, however, was replaced with less costly and morereadily available plant-based and trans-fatty acids. When off the shelfdairy products were tested in the current study, heptadecanoic acid washighest in butter and whole fat yogurt and absent in nonfat dairyproducts. Interestingly, despite consumers movement away from high fatfoods, dairy consumption in humans has been associated with multiplehealth benefits, including lower risks of insulin resistance syndrome,metabolic syndrome, and type 2 diabetes. To date, the mechanism of thebenefits of dairy products on human metabolism has not been determined.Based upon the results using the methods of the present invention, itcan be proposed that heptadecanoic acid may be a key player in themetabolic benefits of dairy products in humans.

To take advantage of these benefits, heptadecanoic acid can be used inacid in a supplement, food additive, food fortifier, beverage additive,beverage fortifier, or pharmaceutical in any form, including as atablet, encapsulated pill, gelcap pill, liquid suspension, spray, andpowder. Additionally, diagnostic tests and assays for heptadecanoic acidin human and animal samples (including blood (serum, plasma, anderythrocyte membranes), urine, and feces) can be used to detect lowheptadecanoic acid levels and to continually monitor heptadecanoic acidlevels in patients. The use of heptadecanoic acid can prevent, stem, andtreat: 1) Elevated ferritin and associated complications, including ironoverload, metabolic syndrome, type 2 diabetes, autoimmune diseases, andneurodegenerative diseases (including but not limited to Alzheimer'sdisease, Parkinson's disease, and restless leg syndrome); and, 2)Metabolic syndrome components and associated complications, includingdyslipidemia, hypertriglyceridemia, elevated glucose, elevated insulin,type 2 diabetes, heart disease, and stroke. These egregious healtheffects can be prevented not only in dolphins, but because of thesimilarities in blood panels, they can be prevented in human mammals aswell.

Referring now to FIG. 15, scatter plots of heptadecanoic acid versusinsulin for both the control populations and the study populations isshown. As shown in FIG. 15, using a proposed therapeutic threshold ofserum heptadecanoic of 0.4 percent as percent of the total fatty acid inserums, can maintain a low insulin (as defined above) level.

It is unknown precisely how high ferritin increases the risk ofdiabetes, but proposed mechanisms include direct injury to the liver andpancreas from excessive deposition or indirect injury from increasedoxidative radicals. Currently, the most accepted means of treatinghyperferritinemia and associated iron overload in humans is phlebotomy(removal of iron in the blood). The methods according to severalembodiments describe methods wherein hyperferritinemia that isassociated with prediabetes can be reversible using a modified diet mostlikely involving increased dietary intake of heptadecanoic acid.Reversal of hyperferritinemia by week 3 using the modified diet wasfollowed by normalization of prediabetes/metabolic syndrome (normalizedglucose, insulin, and triglycerides) at week 24, as described above. Infact, the methods of the present invention can be used to treathyperferritinemia with requiring phlebotomy.

Referring now to FIG. 16, scatter plots of heptadecanoic acid versusferritin levels for both the control populations and the studypopulations is shown. As shown in FIG. 16, using a proposed therapeuticthreshold of serum heptadecanoic of 0.4 percent as percent of the totalfatty acid in serums, can also maintain a therapeutic ferritin (asdefined above) level.

There are several limitations to the current study. First, studydolphins from MMP and Sarasota Bay live in the open ocean, and knowndietary intake was limited to fish fed to MMP dolphins. MMP dolphinslive in netted enclosures in San Diego Bay, and changing populations oflocal fish are readily available to eat. While MMP dolphins can eatlocal fish, however, observation of feeding behaviors by MMP's animalcare staff and maintained dolphin appetites for fed fish support thatthe majority of dietary fish are those that are fed by the MMP.Reference dolphins in the same population and environment, however, didnot have the same decreases in serum ferritin and normalization ofglucose and triglycerides. Second, the proposed direct effect ofheptadecanoic acid on lowering high serum ferritin has to be interpretedwith caution because the feeding study involved fish with higherheptadecanoic acid. The potential impact (or cumulative impacts) ofother nutrients in the modified diet on serum ferritin cannot be ruledout until feeding studies are limited to a heptadecanoic acidsupplement. Identification of 1) higher serum percent heptadecanoic acidas an independent predictor of lower serum ferritin, 2) demonstratedincreased dietary intake and percent serum heptadecanoic acid during thefeeding study, and 3) coincident decreases in serum ferritin andincreases in percent serum heptadecanoic acid by week 3, however,provide evidence that increasing dietary heptadecanoic acid contributedto decreased serum ferritin.

In conclusion, this study with dolphins is the first to proposeheptadecanoic acid deficiencies as a means to detect a risk of or causefor metabolic syndrome and associated hyperferritinemia. Further,dietary supplementation with heptadecanoic acid may help resolve bothconditions. Future research with human populations is needed to assesssimilar relationships between heptadecanoic acid, serum ferritin,metabolic syndrome, and type 2 diabetes. Further, givenhyperferritinemia's association with autoimmunity in humans, thisinvention (use of heptadecanoic acid deficiency detection and resolutionas a means to prevent or manage disease) could also apply to type 1diabetes, an autoimmune disease. Successful demonstration of these linksin humans would support our hypothesis that widespread movement awayfrom high fat dairy products in some countries and decreasingfrequencies of fish meals in other countries have led to heptadecanoicacid deficiencies globally, which may be contributing to an increasingprevalence of hyperferritinemia, metabolic syndrome, and the diabetespandemic.

The use of the terms “a” and “an” and “the” and similar references inthe context of describing the invention (especially in the context ofthe following claims) is to be construed to cover both the singular andthe plural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising”, “having”, “including” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. The use of diagnostic tests and assays forheptadecanoic acid in human and animal samples to detect lowheptadecanoic acid levels and to continually monitor heptadecanoic acidlevels in patients.
 2. The method of claim 1, wherein said samples areselected from the group consisting of serum, plasma, erythrocytemembranes, urine, and feces.